1. Field of the Invention
The present invention relates to a marker for detecting and measuring free radical damage; a method for the direct detection and measurement of the damaging activity of free radicals in vivo is provided.
2. Discussion of the Background
Free radicals are atoms or groups of atoms with an odd (unpaired) number of electrons and can be formed when oxygen interacts with certain molecules. Once formed these highly reactive radicals can start a chain reaction, like dominoes. Their chief danger comes from the damage they can do when they react with important cellular components such as DNA, or the cell membrane. Cells may function poorly or die if this occurs.
A free radical is any molecular species capable of independent existence, that contains one or more unpaired valence electrons not contributing to intramolecular bonding, and isxe2x80x94in that sensexe2x80x94xe2x80x9cfreexe2x80x9d. Free radicals are produced by oxidation/reduction reactions in which there is a transfer of only one electron at a time, or when a covalent bond is broken and one electron from each pair remains with each atom. Free radicals are highly reactive, owing to the tendency of electrons to pairxe2x80x94that is, to pair by the receipt of an electron from an appropriate donor or to donate an electron to an appropriate acceptor. Thus, once formed, free radicals initiate a chain reaction, like dominoesxe2x80x94whenever a free radical reacts with a non-radical, a chain reaction is initiated until two free radicals react and then terminate the propagation with a 2-electron bond (each radical contributing its single unpaired electron).
In biological systems free radicals have a range of transitory existences depending upon their reactivity. Some are stable, e.g. melanins can have a long lifetime, moderately stable ones such as nitric oxide can have lifetimes of xcx9c5 seconds and highly unstable ones such as hydroxyl radicals exist for only a hundredth of a microsecond. The chief danger of free radicals is the damage they can do when they react with important cellular components such as DNA, or the cell membrane. The result on cells of action by free radicals can be diminished or impaired cellular functioning, or even death. For instance, oxygen-free radicals are believed to play a significant role in the aging process. These free radicals often take an electron away from a xe2x80x9ctargetxe2x80x9d molecule to pair with their single free electron. This process is referred to as xe2x80x9coxidationxe2x80x9d and is a known cause of cellular damage and death. Oxygen-free radicals are also implicated in many diseases including neurodegenerative diseases (ALS, Parkinson""s, Alzheimer""s), cataractogenesis, atherosclerosis, diabetes mellitus, ischemia-reperfusion injury, kwashiorkor, and certain toxicities, to mention only a few.
There are many sources of free radicals both within and external to cells. Free radicals are produced by normal ongoing metabolism, especially from the electron transport system in the mitochondria and from a number of normally functioning enzymes. Examples of naturally produced free radicals are: xanthine oxidase, cytochrome p450, monoamine oxidase, and nitric oxide synthase. In the brain, free radicals are produced from the autoxidation of norepinephrine and dopamine. The autoxidation of catechols to quinones generates reduced forms of molecular oxygen, sources of free radicals (e.g., superoxide and hydrogen peroxide). One study suggests that oxidants generated by mitochondria are the major source of oxidative lesions that accumulate with age. See Ames, B. N., M. K. Shigenaga and T. M. Hagen, xe2x80x9cOxidants, antioxidants, and the degenerative diseases of aging.xe2x80x9d Proc. Natl. Acad. Sci. USA 90: 7915-7922 (1993).
Free radicals also function beneficially in normal physiology, including information processing in the brain. Since free radicals can donate an electron to an appropriate acceptor (xe2x80x9creduction reactionxe2x80x9d) or pair their unpaired electron by taking one from an appropriate donor (xe2x80x9coxidation reactionxe2x80x9d) they have major influences on the so-called xe2x80x9credox statexe2x80x9d in cellsxe2x80x94important in normal physiological regulatory reactions. Major free radical targets are molecular complexes that readily give up or acquire a single electron, e.g., those with sulfhydryl/disulfides or with paramagnetic metals (iron, copper). It is calculated that endogenously generated oxygen free radicals make about 10,000 oxidative interactions with DNA per human cell per day (Ames et al., 1993, supra).
Under normal conditions the damaging actions of oxygen free radicals are minimized by abundant protective and repair mechanisms that cells possess, including many enzymes (e.g. superoxide dismutase, catalase) and redox active molecules (e.g., glutathione, thioredoxin).
There is currently an overwhelming need for a sensitive test of free radical damage. For instance, it has been found that the DNA in breast cancer tumors that have generated metastases contain more than twice the amount of free radical damage than tumors that remained confined to the breast. The ability to detect this free radical damage would allow identification of an identifiable metastatic pattern or xe2x80x9cprofile,xe2x80x9d which would be of great benefit in determining if newly diagnosed breast cancer was likely to spread and whether aggressive radiation and chemotherapy is needed. See, e.g., Malins D. et al., xe2x80x9cProgression of Human Breast Cancers to the Metastatic State Is Linked to Hydroxyl Radical-induced DNA Damage,xe2x80x9d Proc. Natl. Acad. Sci. USA 93:2557-2563 (1996). However, no viable test exists.
There currently exists several limited methods for detection of the damaging activity of free radicals in the body. One method uses an isoprostane called IPF2alpha-I, an abundant and stable byproduct of free-radical catalyzed oxidation of arachidonic acid, which is easily detected in urine. Arachidonic acid is a fatty molecule found in cell membranes throughout the body. Pratico D. et al., xe2x80x9cLocalization of distinct F2-isoprostanes in human atherosclerotic lesions,xe2x80x9d J Clin. Invest. 100(8):2028-2034 (1997). Other methods take advantage of the formation of carbonyls from lipids, proteins, carbohydrates, and nucleic acids during oxidative stress. For example, metal-catalyzed, xe2x80x9csite-specificxe2x80x9d oxidation of several amino acid side-chains has been reported to result in the production of aldehydes or ketones, and peroxidation of lipids to generate reactive aldehydes such as malondialdehyde and hydroxynonenal. These oxidative changes have been detected in situ using 2,4-dinitrophenylhydrazine labeling linked to an antibody system specific to localized biomacromolecule-bound carbonyl reactivity. See Smith, M. A. et al., xe2x80x9cCytochemical demonstration of oxidative damage in Alzheimer disease by immunochemical enhancement of the carbonyl reaction with 2,4-dinitrophenylhydrazine,xe2x80x9d J Histochem. Cytochem. 46(6):731-735 (1998). Use of immunochemical assays for detection of carbonyl moieties resulting from oxidative damage to bovine serum albumin has also been reported. See Keller, R. J. et al., xe2x80x9cImmunochemical detection of oxidized proteins,xe2x80x9d Chem. Res. Toxicol. 6(4): 430-433 (1993), and Mateos-Nevado, D. J., xe2x80x9cImmunological detection and quantification of oxidized proteins by labeling with digoxigenin,xe2x80x9d Biosci. Biotechnol. Biochem. 62(3):419-423 (1998).
These methods, however, are limited in their usefulness and applicability due to the highly specific and system-limited nature of the markers utilized for detection. The present invention, in contrast, provides a marker for the existence and detection/measurement of free radical damage which is highly sensitive and present in a majority of human fluids and tissues.
The need for rapid, immediate and continuous detection of free radical damage, locally or systemically, is met by the present invention. The present invention provides a marker for the existence and detection of free radical damage.
The marker may be used as a xe2x80x9cbiochemical tag,xe2x80x9d thereby allowing for sensitive detection and measurement of the efficacy of clinical drugs and therapeutics which result in the generation of free radicals, such as Photofrin(copyright) (porfimer sodium), or which act to limit free-radical damage. The marker also acts as a xe2x80x9cbiological tagxe2x80x9d of a process implicated in a wide array of diseases and sequelae and, accordingly, may be used to test for the occurrence or non-occurrence of such diseases and sequelae. One such disease is ischemia.
Additional advantages, applications, embodiments and variants of the invention are included in the Detailed Description of the Invention and Examples sections.
As used herein, the term xe2x80x9cischemic event,xe2x80x9d and xe2x80x9cischemic statexe2x80x9d mean that the patient has experienced a local and/or temporary ischemia due to partial or total obstruction of the blood circulation to an organ. Additionally, the following abbreviations are utilized herein to refer to the following amino acids:
A separate test method for ischemia using the marker of the present invention is described by the inventors herein in pending U.S. patent applications Ser. Nos. 09/165,581 and 09/165,926 (both titled xe2x80x9cTEST FOR RAPID EVALUATION OF ISCHEMIC STATES AND KIT), filed Oct. 2, 1998, by the inventors herein (Bar-Or and Lau) in conjunction with inventor J. Winkler, both of which are herein incorporated by reference in their entirety.
The present invention teaches a marker useful for detection and measurement of free radical damage. Specifically, the invention takes advantage of alterations which occur to the N-terminus of the albumin molecule, a circulating protein in human blood, in the presence of free radicals. These alterations effect the ability of the N-terminus of the albumin molecule to bind metals.
Human serum albumin (xe2x80x9cHSAxe2x80x9d) is the most abundant protein in blood (40 g/1) and the major protein produced by the liver. Many other body fluids also contain albumin. The main biological function of albumin is believed to be regulation of the colloidal osmotic pressure of blood. The amino acid and structure of human albumin have been determined. Specifically, human albumin is a single polypeptide chain consisting of 585 amino acids folded into three homologous domains with one free sulfhydryl group on residue #34, having a specific amino acid content as follows:
Amino Acids: Asp Asn Thr Ser Glu Gln Pro Gly Ala Cys Val Met Ile Leu Tyr Phe His Lys Trp Arg
Residues 39 15 30 22 60 23 25 12 63 35 39 6 8 61 18 30 16 58 1 23
The N-terminus of albumin is known to possess metal binding capacity (see Chan et al., xe2x80x9cSite-specific N-terminal auto-degradation of human serum albumin,xe2x80x9d Eur. J Biochem. 277, 524-528 (1995)), and damage to the structure of albumin resulting from the binding of metals such as vanadium, copper and iron has been detected by changes to the thiol groups and the tryptophan residue (Quinlan et al., xe2x80x9cVanadium and copper in clinical solutions of albumin and their potential to damage protein structure,xe2x80x9d J Pharm. Sci., 81, 611-614 (1992)). Additionally, oxidative modifications to structure of bovine serum albumin have been noted generally, but not within the N-terminus as in the present invention. See Davies et al., xe2x80x9cProtein damage and degradation by oxygen radicals,xe2x80x9d J Biol. Chem. 262, 9895-9901, 9908-9913 (1987); Marx, G. et al., xe2x80x9cSite-specific modification of albumin by free radicals. Reaction with copper(II) and ascorbate,xe2x80x9d Biochem. J 236(2):397-400 (1986). The present invention takes advantage of the discovery by the present inventors of the previously unknown phenomenon that oxidative modifications to the structure of human serum albumin occur within the N-terminus such that metal binding within the site is effectively precluded.
While not being bound by any particular theory, it is believed that a combination of two separate phenomena explain the mechanism by which the presence of free radicals causes an alteration of the N-terminus of albumin resulting in a loss of metal-binding capacity. First, it is believed that the alteration may be due to the loss of a proton on the backbone of the alanine residue causing cyclization of the aspartate carboxyl group with the alanine carbon. (Cyclization such as this one has been reported previously for albumin, as an autodegradation process with heat at 57 degrees Celsius. See Chan et al., 1995, supra.) Applicants, believe that the cyclization process is accelerated in the presence of free radicals. Second, it is possible that free radicals cause the clipping of the two amino terminals (aspartate-alanine) from the N-terminal of albumin and the cleaved dipeptide undergoes cyclization. Regardless of the underlying mechanism, the effect of free radicals on the N-terminus of albumin is discovered to be a loss of metal binding capacity.
The occurrence or non-occurrence of the marker may be detected by the method comprising the steps of (a) contacting a biological sample containing albumin with an excess quantity of a metal ion salt, said metal ion capable of binding to the N-terminus of naturally occurring human albumin, to form a mixture containing bound metal ions and unbound metal ions, (b) determining the amount of bound metal ions, and (c) correlating the amount of bound metal ions to a known value to determine the occurrence or non-occurrence of the marker of the present invention. In this method, said excess quantity of metal ion salt may comprise a predetermined quantity and the quantity of unbound metal ions may be detected to determine the amount of bound metal ions. Additionally, the compound selected from the group consisting of Asp-Ala-His-Lys-R [SEQ ID NO:1], wherein R is any chemical group capable of being detected when bound to any compound capable of binding to the N-terminus of naturally occurring human albumin, may be utilized to facilitate detection.
Preferred embodiments of the first method include samples of serum or plasma, or purified albumin. Preferred embodiments also include use of a metal ion salt comprising a salt of a transition metal ion of Groups 1 b-7b or 8 of the Periodic Table of the elements, a metal selected from the group consisting of V, As, Co, Sb, Cr, Mo, Mn, Ba, Zn, Ni, Hg, Cd, Fe, Pb, Au and Ag, or cobalt. Also preferred is detection of the amount of bound metal ions (or, in the case where the excess quantity of metal ion salt is a predetermined quantity, detection of the quantity of unbound metal ions) by atomic absorption or atomic emission spectroscopy or immunological assay. These detection mechanisms are also preferred for determination of the quantity of the compound Asp-Ala-His-Lys-R [SEQ ID NO:1] which is complexed with the metal ion salt in order to detect the quantity of unbound metal ions. A preferred method for conducting said immunological assay is using an antibody specific to an antigen comprising the compound Asp-Ala-His-Lys-R [SEQ ID NO:1], wherein R is said metal ion.
A second method of the present invention for detecting the occurrence or non-occurrence of the marker of the present invention comprises the steps of: (a) contacting a biological sample containing albumin with a predetermined excess quantity of a salt of a metal selected from the group consisting of V, As, Co, Sb, Cr, Mo, Mn, Ba, Zn, Ni, Hg, Cd, Fe, Pb, Au and Ag, to form a mixture containing bound metal ions and unbound metal ions, (b) contacting said mixture with an aqueous color forming compound solution to form a colored solution, wherein said compound is capable of forming color when bound to said metal ion, (c) determining the color intensity of said colored solution to detect the presence of unbound metal ions to provide a measure of bound metal ions, and (d) correlating the amount of bound metal ions to a known value to determine the occurrence or non-occurrence of the marker of the present invention. Preferred embodiments of this method include the additional step of diluting said colored solution with an aqueous solution isosmotic with blood serum or plasma prior to step (c). Also preferred are: using ferrozine as the color forming compound, and, alternatively, using the compound Asp-Ala-His-Lys-R [SEQ ID NO:1], wherein R is any group capable of forming color when bound to said metal ion as the aqueous color forming compound. Conducting steps (b) and (c) in a pH range of 7 to 9 is preferred. Further, conducting steps (b) and (c) using a spectrophotometer is preferred. Preferred samples in this method also comprise serum, plasma, or purified albumin. A preferred metal ion salt is cobalt.
A third method of the present invention for detecting the occurrence or non-occurrence of the marker of the present invention comprises the steps of: (a) detecting the amount of copper ions present in a purified albumin sample, and (b) correlating the quantity of copper ions present with a known value to determine the occurrence or non-occurrence of the marker of the present invention. Preferred methods for detection of the amount of copper ions present in the purified albumin sample are by atomic absorption, atomic emission spectroscopy and immunological assay. A preferred method of conducting said immunological assay uses an antibody specific to an antigen comprising the compound Asp-Ala-His-Lys-R [SEQ ID NO:1], wherein R is copper.
A fourth method of the present invention for detecting the occurrence or non-occurrence of the marker of the present invention comprises the steps of: (a) contacting a purified albumin sample of said patient, with an aqueous color forming compound solution to form a colored solution, wherein said compound is capable of forming color when bound to copper, (b) determining the color intensity of said colored solution to determine the amount copper in said sample, and (c) correlating the amount of copper to a known value to determine the occurrence or non-occurrence of the marker of the present invention. Preferred embodiments of this method include the additional step of diluting said colored solution with an aqueous solution isosmotic with blood serum or plasma prior to step (b). Also preferred are: using ferrozine as the color forming compound, and, alternatively, using the compound Asp-Ala-His-Lys-R [SEQ ID NO:1], wherein R is any group capable of forming color when bound to copper ion as the aqueous color forming compound. Conducting steps (a) and (b) in a pH range of 7 to 9 is preferred. Further, conducting step (b) using a spectrophotometer is preferred.
A method of the present invention uses the marker to assess the clinical efficacy of photosensitizing agents (and light-activated compounds) used in photodynamic therapy for the treatment of tumors, such as Photofrin(copyright) (porfimer sodium). In this procedure, a photosensitizing chemotherapeutic agent is injected into the blood stream, followed by application of low energy, non-thermal laser light 40 to 50 hours after injection, directly to the vicinity of the tumor. The laser light in combination with the photosensitizing agent, which is retained at much higher concentrations in the tumor tissue than normal tissue, causes destruction of the tumor cells. Normal tissues remain virtually untouched. Detection of the existence of the marker, and a corresponding change in the level thereof during the application of the laser, provides a indication (and allows for quantitation) of the effectiveness of the photodynamic therapy. Several methods allow for detection and measurement of the marker. For instance, a metal ion salt may be mixed with a purified albumin sample obtained from a patient serum, plasma, fluid or tissue sample. The metal ion salt will not bind with the marker due to the alteration of the binding site of the N-terminus. Accordingly, the existence and concentration of the marker can be determined by detection of the presence and quantity of bound or unbound metal ion. Measurement can be conducted by atomic absorption, infrared spectroscopy, high-performance liquid chromatography (xe2x80x9cHPLCxe2x80x9d) or other standard or non-standard methods, including radioactive immunoassay techniques.
A greater understanding of the present invention and of its many advantages may be had from the following examples, given by way of illustration. The following examples are illustrative of some of the methods, applications, embodiments and variants of the present invention. They are, of course, not to be considered in any way limitative of the invention. Numerous changes and modification can be made with respect to the invention with departing from the spirit or scope of the invention.
An in vitro experimental model was conducted using an octapeptide having the same eight amino acid sequence as the N-terminus of human albumin. The following preliminary experiments (Nos. 1 through 11) demonstrate the properties and critical characteristics of the peptide probe.